Three objectives form the basis for this study of drug hydroxylation: 1. To characterize microsomal enzyme mechanisms responsible for PNP hydroxylation, especially with regard to the observed activation by Vitamin C in vitro. Nitrocatechol (NTC) has been identified as a product of both p-nitroanisole (PNA) and p-nitrophenol (PNP) metabolism by rat liver. The metabolic system is localized in liver microsomes and has characteristics similar to those of a mixed function oxidase. Products of the reaction (PNA yields PNP yields NTC) are further metabolized to glucuronide and sulfate conjugates. The initial hydroxylation of PNP can be activated or inhibited in vivo (phenobarbital, SKF525A) and in vitro (Vitamin C, cysteine, ATP, SH-group inhibitors). A role for Vitamin C in the electron transport mechanism for drug hydroxylation has recently been found by us. The reaction is dependent on Vitamin C plus Microsomes plus NADPH plus O2. The enzymic mechanism for PNP hydrolation will be defined and applicability of the mechanism to clinically useful drugs (e.g. diphenylhydantoin) tested. 2. To study chemical mechanisms for drug hydroxylation (by using stable isotopes (O18)) and the possible role of peroxides and epoxides and other intermediates in the hydroxylation of PNP. The mechanism of aromatic drug oxidation will be studied by using stable isotopes (P18, H2O18) with product analysis by mass spectrometry. 3. To demonstrate a limiting effect of glucuronide conjugation on the rate of aromatic hydroxylation of PNP, diphenylhydantoin (DPH) and clinically useful drugs which have a high dependency on metabolic clearance by hydroxylation and conjugation. The initial speed of PNP hydroxylation can be sustained by addition of a glucuronide forming system. Product inhibition can thus be obviated by conjugation of the product restoring first order kinetics to the reaction. The overall purpose of this study is to investigate the hydroxylation of aromatic drugs and the conjugation of their corresponding phenols and catechols.